Abstract
Background & Aims: Liver fibrosis is a pathological healing process resulting from hepatic stellate cell (HSC) activation and the generation of myofibroblasts from activated HSCs. The precise underlying mechanisms of liver fibrogenesis are still largely vague due to lack of understanding the functional heterogeneity of activated HSCs during liver injury. Approach and Results: In this study, to define the mechanism of HSC activation, we performed the transcriptomic analysis at single-cell resolution (scRNA-seq) on HSCs in mice treated with carbon tetrachloride (CCl4). By employing LRAT-Cre:Rosa26mT/mG mice, we were able to isolate an activated GFP-positive HSC lineage derived cell population by fluorescence-activated cell sorter (FACS). A total of 8 HSC subpopulations were identified based on an unsupervised analysis. Each HSC cluster displayed a unique transcriptomic profile, despite all clusters expressing common mouse HSC marker genes. We demonstrated that one of the HSC subpopulations expressed high levels of mitosis regulatory genes, velocity, and monocle analysis indicated that these HSCs are at transitioning and proliferating phases at the beginning of HSCs activation and will eventually give rise to several other HSC subtypes. We also demonstrated cell clusters representing HSC-derived mature myofibroblast populations that express myofibroblasts hallmark genes with unique contractile properties. Most importantly, we found a novel HSC cluster that is likely to be critical in liver regeneration, immune reaction, and vascular remodeling, in which the unique profiles of genes such as Rgs5, Angptl6, and Meg3 are highly expressed. Lastly, we demonstrated that the heterogeneity of HSCs in the injured mouse livers is closely similar to that of cirrhotic human livers. Conclusions: Collectively, our scRNA-seq data provided insight into the landscape of activated HSC populations and the dynamic transitional pathway from HSC to myofibroblasts in response to liver injury.
Highlights
Liver cirrhosis is a major health burden in patients with liver diseases
To investigate the heterogeneity of activated hepatic stellate cell (HSC) during liver fibrogenesis, we performed scRNA-seq analysis on HSCs in mice treated with carbon tetrachloride (CCl4 )
In HSCs, Cre expression driven by Lecithin retinol acyltransferase (LRAT) promoter will lead to HSCs and their derived cell population to have GFP expression to replace red tdTomato fluorescence (Figure 1A, upper panel)
Summary
Liver cirrhosis is a major health burden in patients with liver diseases. It frequently occurs following chronic insults to trigger wound healing responses in the liver and induces the excessive deposition of extracellular matrix (ECM) [1]. HSCs reduce the expression of genes such as peroxisome proliferator-activated receptor gamma (PPARγ) [3], lose lipid droplets, and activate into myofibroblasts by starting to express genes that encode contractile proteins and extracellular fibrogenic genes such alpha-smooth muscle actin (α-SMA) and collagen Type I, respectively Upregulation of these genes is critical for wound healing in the reparative processes [1]. HSCs play other essential roles such as in the release of different kinds of cytokines (Stellakines) to regulate inflammation, vasoactive response, angiogenesis, and promote migration of other hepatic lineage cells to the injury site for the reconstruction of the liver architecture and function [1] Whether these different roles are fulfilled by a single and homogeneous population of activated HSCs, or by different sublineage cells with diversified function remains elusive. Our analysis further provides a detailed landscape of HSC transformation and uncovered previously unknown HSC subtypes and their functional heterogeneity, and further shed light on the dynamics of HSC-to-myofibroblasts transition in response to liver injury
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